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1
Siththanandan, Verl B., and James R. Sellers. "Regulation of myosin 5a and myosin 7a." Biochemical Society Transactions 39, no. 5 (September 21, 2011): 1136–41. http://dx.doi.org/10.1042/bst0391136.
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The myosin superfamily is diverse in its structure, kinetic mechanisms and cellular function. The enzymatic activities of most myosins are regulated by some means such as Ca2+ ion binding, phosphorylation or binding of other proteins. In the present review, we discuss the structural basis for the regulation of mammalian myosin 5a and Drosophila myosin 7a. We show that, although both myosins have a folded inactive state in which domains in the myosin tail interact with the motor domain, the details of the regulation of these two myosins differ greatly.
2
Baines, I. C., H. Brzeska, and E. D. Korn. "Differential localization of Acanthamoeba myosin I isoforms." Journal of Cell Biology 119, no. 5 (December 1, 1992): 1193–203. http://dx.doi.org/10.1083/jcb.119.5.1193.
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Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.
3
Berg, Jonathan S., Bradford C. Powell, and Richard E. Cheney. "A Millennial Myosin Census." Molecular Biology of the Cell 12, no. 4 (April 2001): 780–94. http://dx.doi.org/10.1091/mbc.12.4.780.
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The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans,Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana,Saccharomyces cerevisiae, Schizosaccharomyces pombe, andDictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms.
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Hammer, J. A., B. Bowers, B. M. Paterson, and E. D. Korn. "Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail." Journal of Cell Biology 105, no. 2 (August 1, 1987): 913–25. http://dx.doi.org/10.1083/jcb.105.2.913.
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We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.
5
Heintzelman, M. B., T. Hasson, and M. S. Mooseker. "Multiple unconventional myosin domains of the intestinal brush border cytoskeleton." Journal of Cell Science 107, no. 12 (December 1, 1994): 3535–43. http://dx.doi.org/10.1242/jcs.107.12.3535.
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Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.
6
Berg, J. S., B. H. Derfler, C. M. Pennisi, D. P. Corey, and R. E. Cheney. "Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin." Journal of Cell Science 113, no. 19 (October 1, 2000): 3439–51. http://dx.doi.org/10.1242/jcs.113.19.3439.
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Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.
7
Post, P. L., G. M. Bokoch, and M. S. Mooseker. "Human myosin-IXb is a mechanochemically active motor and a GAP for rho." Journal of Cell Science 111, no. 7 (April 1, 1998): 941–50. http://dx.doi.org/10.1242/jcs.111.7.941.
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The heavy chains of the class IX myosins, rat myr5 and human myosin-IXb, contain within their tail domains a region with sequence homology to GTPase activating proteins for the rho family of G proteins. Because low levels of myosin-IXb expression preclude purification by conventional means, we have employed an immunoadsorption strategy to purify myosin-IXb, enabling us to characterize the mechanochemical and rho-GTPase activation properties of the native protein. In this report we have examined the light chain content, actin binding properties, in vitro motility and rho-GTPase activity of human myosin-IXb purified from leukocytes. The results presented here indicate that myosin-IXb contains calmodulin as a light chain and that it binds to actin with high affinity in both the absence and presence of ATP. Myosin-IXb is an active motor which, like other calmodulin-containing myosins, exhibits maximal velocity of actin filaments (15 nm/second) in the absence of Ca2+. Native myosin-IXb exhibits GAP activity on rho. Class IX myosins may be an important link between rho and rho-dependent remodeling of the actin cytoskeleton.
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Wylie, Steven R., and Peter D. Chantler. "Myosin IIC: A Third Molecular Motor Driving Neuronal Dynamics." Molecular Biology of the Cell 19, no. 9 (September 2008): 3956–68. http://dx.doi.org/10.1091/mbc.e07-08-0744.
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Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells.
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O'Halloran, T. J., S. Ravid, and J. A. Spudich. "Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation." Journal of Cell Biology 110, no. 1 (January 1, 1990): 63–70. http://dx.doi.org/10.1083/jcb.110.1.63.
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The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.
10
Hasson, T., and M. S. Mooseker. "Porcine myosin-VI: characterization of a new mammalian unconventional myosin." Journal of Cell Biology 127, no. 2 (October 15, 1994): 425–40. http://dx.doi.org/10.1083/jcb.127.2.425.
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We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC-PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.
11
Pernier, Julien, and Kristine Schauer. "Does the Actin Network Architecture Leverage Myosin-I Functions?" Biology 11, no. 7 (June 29, 2022): 989. http://dx.doi.org/10.3390/biology11070989.
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The actin cytoskeleton plays crucial roles in cell morphogenesis and functions. The main partners of cortical actin are molecular motors of the myosin superfamily. Although our understanding of myosin functions is heavily based on myosin-II and its ability to dimerize, the largest and most ancient class is represented by myosin-I. Class 1 myosins are monomeric, actin-based motors that regulate a wide spectrum of functions, and whose dysregulation mediates multiple human diseases. We highlight the current challenges in identifying the “pantograph” for myosin-I motors: we need to reveal how conformational changes of myosin-I motors lead to diverse cellular as well as multicellular phenotypes. We review several mechanisms for scaling, and focus on the (re-) emerging function of class 1 myosins to remodel the actin network architecture, a higher-order dynamic scaffold that has potential to leverage molecular myosin-I functions. Undoubtfully, understanding the molecular functions of myosin-I motors will reveal unexpected stories about its big partner, the dynamic actin cytoskeleton.
12
Shu, S., R. J. Lee, J. M. LeBlanc-Straceski, and T. Q. Uyeda. "Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium." Journal of Cell Science 112, no. 13 (July 1, 1999): 2195–201. http://dx.doi.org/10.1242/jcs.112.13.2195.
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Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: Mydelta824-941, Mydelta943-1464, Mydelta943-1194 and Mydelta1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for Mydelta943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow.
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Liu, Xiong, Shi Shu, and Edward D. Korn. "Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro." Proceedings of the National Academy of Sciences 117, no. 27 (June 22, 2020): 15666–72. http://dx.doi.org/10.1073/pnas.2001892117.
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Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail–head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.
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Holmes, K. C., D. R. Trentham, R. Simmons, and Peter G. Gillespie. "Myosin I and adaptation of mechanical transduction by the inner ear." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 359, no. 1452 (December 29, 2004): 1945–51. http://dx.doi.org/10.1098/rstb.2004.1564.
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Twenty years ago, the description of hair-cell stereocilia as actin-rich structures led to speculation that myosin molecules participated in mechanical transduction in the inner ear. In 1987, Howard and Hudspeth proposed specifically that a myosin I might mediate adaptation of the transduction current carried by hair cells, the sensory cells of the ear. We exploited the myosin literature to design tests of this hypothesis and to show that the responsible isoform is myosin 1c. The identification of this myosin as the adaptation motor would have been impossible without thorough experimentation on other myosins, particularly muscle myosins. The sliding-filament hypothesis for muscle contraction has thus led to a detailed understanding of the behaviour of hair cells.
15
Lee, Kyoung Hwan, Guidenn Sulbarán, Shixin Yang, Ji Young Mun, Lorenzo Alamo, Antonio Pinto, Osamu Sato, et al. "Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals." Proceedings of the National Academy of Sciences 115, no. 9 (February 14, 2018): E1991—E2000. http://dx.doi.org/10.1073/pnas.1715247115.
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Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head–head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head–head/head–tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.
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Redowicz, Maria Jolanta. "Myosins and pathology: genetics and biology." Acta Biochimica Polonica 49, no. 4 (December 31, 2002): 789–804. http://dx.doi.org/10.18388/abp.2002_3739.
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This article summarizes current knowledge on the genetics and possible molecular mechanisms of Human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (beta cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the beta cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments.
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Petersen, Karl J., Holly V. Goodson, Ashley L. Arthur, G. W. Gant Luxton, Anne Houdusse, and Margaret A. Titus. "MyTH4-FERM myosins have an ancient and conserved role in filopod formation." Proceedings of the National Academy of Sciences 113, no. 50 (November 23, 2016): E8059—E8068. http://dx.doi.org/10.1073/pnas.1615392113.
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The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.
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Miller, D. D., S. P. Scordilis, and P. K. Hepler. "Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata." Journal of Cell Science 108, no. 7 (July 1, 1995): 2549–63. http://dx.doi.org/10.1242/jcs.108.7.2549.
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The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.
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McCaffrey, Mary W., and Andrew J. Lindsay. "Roles for myosin Va in RNA transport and turnover." Biochemical Society Transactions 40, no. 6 (November 21, 2012): 1416–20. http://dx.doi.org/10.1042/bst20120172.
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Mammals express three class V myosins. Myosin Va is widely expressed, but enriched in the brain, testes and melanocytes, myosin Vb is expressed ubiquitously, and myosin Vc is believed to be epithelium-specific. Myosin Va is the best characterized of the three and plays a key role in the transport of cargo to the plasma membrane. Its cargo includes cell-surface receptors, pigment and organelles such as the endoplasmic reticulum. It is also emerging that RNA and RNA-BPs (RNA-binding proteins) make up another class of myosin Va cargo. It has long been established that the yeast class V myosin, Myo4p, transports mRNAs along actin cables into the growing bud, and now several groups have reported a similar role for class V myosins in higher eukaryotes. Myosin Va has also been implicated in the assembly and maintenance of P-bodies (processing bodies), cytoplasmic foci that are involved in mRNA storage and degradation. The present review examines the evidence that myosin Va plays a role in the transport and turnover of mRNA.
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Jung, Hyun Suk, Satoshi Komatsu, Mitsuo Ikebe, and Roger Craig. "Head–Head and Head–Tail Interaction: A General Mechanism for Switching Off Myosin II Activity in Cells." Molecular Biology of the Cell 19, no. 8 (August 2008): 3234–42. http://dx.doi.org/10.1091/mbc.e08-02-0206.
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Intramolecular interaction between myosin heads, blocking key sites involved in actin-binding and ATPase activity, appears to be a critical mechanism for switching off vertebrate smooth-muscle myosin molecules, leading to relaxation. We have tested the hypothesis that this interaction is a general mechanism for switching off myosin II–based motile activity in both muscle and nonmuscle cells. Electron microscopic images of negatively stained myosin II molecules were analyzed by single particle image processing. Molecules from invertebrate striated muscles with phosphorylation-dependent regulation showed head–head interactions in the off-state similar to those in vertebrate smooth muscle. A similar structure was observed in nonmuscle myosin II (also phosphorylation-regulated). Surprisingly, myosins from vertebrate skeletal and cardiac muscle, which are not intrinsically regulated, undergo similar head–head interactions in relaxing conditions. In all of these myosins, we also observe conserved interactions between the ‘blocked’ myosin head and the myosin tail, which may contribute to the switched-off state. These results suggest that intramolecular head–head and head-tail interactions are a general mechanism both for inducing muscle relaxation and for switching off myosin II–based motile activity in nonmuscle cells. These interactions are broken when myosin is activated.
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Novakovic, Valerie A., and Gary E. Gilbert. "Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid." Blood 136, no. 21 (November 19, 2020): 2469–72. http://dx.doi.org/10.1182/blood.2020005930.
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Abstract Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine–binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.
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Trivedi, Darshan V., Suman Nag, Annamma Spudich, Kathleen M. Ruppel, and James A. Spudich. "The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches." Annual Review of Biochemistry 89, no. 1 (June 20, 2020): 667–93. http://dx.doi.org/10.1146/annurev-biochem-011520-105234.
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Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human β-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human β-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.
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Jontes, James D., E. Michael Ostap, Thomas D. Pollard, and Ronald A. Milligan. "Three-dimensional Structure of Acanthamoeba castellanii Myosin-IB (MIB) Determined by Cryoelectron Microscopy of Decorated Actin Filaments." Journal of Cell Biology 141, no. 1 (April 6, 1998): 155–62. http://dx.doi.org/10.1083/jcb.141.1.155.
Full textAbstract:
The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a “classical” myosin-I, Acanthamoeba myosin-IB (MIB), at ∼18 Å resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, ∼10°, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.
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Trybus, KM, GS Waller, and TA Chatman. "Coupling of ATPase activity and motility in smooth muscle myosin is mediated by the regulatory light chain." Journal of Cell Biology 124, no. 6 (March 15, 1994): 963–69. http://dx.doi.org/10.1083/jcb.124.6.963.
Full textAbstract:
Smooth muscle myosin acts as a molecular motor only if the regulatory light chain (RLC) is phosphorylated. This subunit can be removed from myosin by a novel method involving the use of trifluoperazine. The motility of RLC-deficient myosin is very slow, but native properties are restored when RLC is rebound. Truncating 6 residues from the COOH terminus of the RLC had no effect on phosphorylated myosin's motor properties, while removal of the last 12 residues reduced velocity by approximately 30%. Very slow movement was observed once 26 residues were deleted, or with myosin containing only the COOH-terminal RLC domain. These two mutants thus mimicked the behavior of RLC-deficient myosin, with the important difference that the mutant myosins were monodisperse when assayed by sedimentation velocity and electron microscopy. The decreased motility therefore cannot be caused by aggregation. A common feature of RLC-deficient myosin and the mutant myosins that moved actin slowly was an increased myosin ATPase compared with dephosphorylated myosin, and a lower actin-activated ATPase than obtained with phosphorylated myosin. These results suggest that the COOH-terminal portion of an intact RLC is involved in interactions that regulate myosin's "on-off" switch, both in terms of completely inhibiting and completely activating the molecule.
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Watabe, Shugo. "Temperature plasticity of contractile proteins in fish muscle." Journal of Experimental Biology 205, no. 15 (August 1, 2002): 2231–36. http://dx.doi.org/10.1242/jeb.205.15.2231.
Full textAbstract:
SUMMARY Three myosin heavy chain isoforms with different actin-activated Mg2+-ATPase activities were found in the fast skeletal muscle from carp (Cyprinus carpio) acclimated to 10 and 30°C. The composition of three types of myosin heavy chain was dependent on acclimation temperature,demonstrating the presence of temperature-specific myosin isoforms in carp. Subsequently, the temperature-dependence of the sliding velocity of fluorescent F-actin in myosins isolated from 10°C- and 30°C-acclimated carp was measured. At 8°C, the filament velocity was three times higher for myosin from 10°C- than from 30°C-acclimated fish. Activation energies (Ea) for the sliding velocity of F-actin were 63 and 111 kJ mol-1 for myosins from 10°C- and 30°C-acclimated fish, respectively. Activation energy for actin-activated Mg2+-ATPase activity was 0.46 kJ mol-1 in myosin from 10°C-acclimated fish and 0.54 kJ mol-1 in myosin from 30°C-acclimated fish. The inactivation rate constant(KD) of Ca2+-ATPase was 7.5×10-4s-1 at 30°C for myosin from 10°C-acclimated fish, which was approximately twice that for myosin from 30°C-acclimated fish. It is suggested that these differences in thermostability reflect a more flexible structure of the myosin molecule in cold-acclimated carp, which results in a reduced activation enthalpy for contraction and, hence, a higher sliding velocity at low temperatures. Structural analysis of cDNAs encoding the carp myosin heavy chain demonstrated striking differences in two surface loops of myosin subfragment-1 (S1), loops 1 and 2, between the 10°C and 30°C types, which were predominantly expressed in carp acclimated to 10°C and 30°C, respectively. Chimeric myosins composed of Dictyostelium discoideum myosin backbones with loop sequences of carp S1 heavy chain isoforms demonstrated that the diversity of the loop 2 sequence of carp S1 affected the Vmax of actin-activated Mg2+-ATPase activity.
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Kurth, Elizabeth G., Valera V. Peremyslov, Hannah L. Turner, Kira S. Makarova, Jaime Iranzo, Sergei L. Mekhedov, Eugene V. Koonin, and Valerian V. Dolja. "Myosin-driven transport network in plants." Proceedings of the National Academy of Sciences 114, no. 8 (January 17, 2017): E1385—E1394. http://dx.doi.org/10.1073/pnas.1620577114.
Full textAbstract:
We investigate the myosin XI-driven transport network inArabidopsisusing protein–protein interaction, subcellular localization, gene knockout, and bioinformatics analyses. The two major groups of nodes in this network are myosins XI and their membrane-anchored receptors (MyoB) that, together, drive endomembrane trafficking and cytoplasmic streaming in the plant cells. The network shows high node connectivity and is dominated by generalists, with a smaller fraction of more specialized myosins and receptors. We show that interaction with myosins and association with motile vesicles are common properties of the MyoB family receptors. We identify previously uncharacterized myosin-binding proteins, putative myosin adaptors that belong to two unrelated families, with four members each (MadA and MadB). Surprisingly, MadA1 localizes to the nucleus and is rapidly transported to the cytoplasm, suggesting the existence of myosin XI-driven nucleocytoplasmic trafficking. In contrast, MadA2 and MadA3, as well as MadB1, partition between the cytosolic pools of motile endomembrane vesicles that colocalize with myosin XI-K and diffuse material that does not. Gene knockout analysis shows that MadB1–4 contribute to polarized root hair growth, phenocopying myosins, whereas MadA1–4 are redundant for this process. Phylogenetic analysis reveals congruent evolutionary histories of the myosin XI, MyoB, MadA, and MadB families. All these gene families emerged in green algae and show concurrent expansions via serial duplication in flowering plants. Thus, the myosin XI transport network increased in complexity and robustness concomitantly with the land colonization by flowering plants and, by inference, could have been a major contributor to this process.
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Yumura, Shigehiko, and Taro Q. P. Uyeda. "Transport of Myosin II to the Equatorial Region without Its Own Motor Activity in Mitotic Dictyostelium Cells." Molecular Biology of the Cell 8, no. 10 (October 1997): 2089–99. http://dx.doi.org/10.1091/mbc.8.10.2089.
Full textAbstract:
Fluorescently labeled myosin moved and accumulated circumferentially in the equatorial region of dividingDictyostelium cells within a time course of 4 min, followed by contraction of the contractile ring. To investigate the mechanism of this transport process, we have expressed three mutant myosins that cannot hydrolyze ATP in myosin null cells. Immunofluorescence staining showed that these mutant myosins were also correctly transported to the equatorial region, although no contraction followed. The rates of transport, measured using green fluorescent protein-fused myosins, were indistinguishable between wild-type and mutant myosins. These observations demonstrate that myosin is passively transported toward the equatorial region and incorporated into the forming contractile ring without its own motor activity.
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Neu, N., K. W. Beisel, M. D. Traystman, N. R. Rose, and S. W. Craig. "Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis." Journal of Immunology 138, no. 8 (April 15, 1987): 2488–92. http://dx.doi.org/10.4049/jimmunol.138.8.2488.
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Abstract Several mouse strains are susceptible to immunopathic myocarditis after infection with Coxsackievirus B3 (CB3). This disease is associated with autoantibodies that are directed against myosin. In this study we characterized sera from CB3-infected mice for their reactivity with three different myosin isoforms (heart, skeletal muscle, and brain myosins) and for autoantibody isotype by using an ELISA. Competitive inhibition assays and absorption studies with various myosins demonstrated the presence of two autoantibody populations in sera of susceptible A.CA and A.SW mice. The first was specific for cardiac myosin and was mainly IgG. The second antibody population cross-reacted with heart, skeletal muscle, and brain myosin and was mainly IgM. B10.PL/SgSf and B10.A/SgSf mice, which do not develop immunopathic myocarditis, produced only the IgM autoantibody population cross-reactive with all three myosin isoforms. Because the heart-specific myosin autoantibodies were found exclusively in the mouse strains that developed immunopathic myocarditis, they can be considered a serologic marker for autoimmune heart disease.
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Rodriguez, Olga C., and Richard E. Cheney. "Human myosin-Vc is a novel class V myosin expressed in epithelial cells." Journal of Cell Science 115, no. 5 (March 1, 2002): 991–1004. http://dx.doi.org/10.1242/jcs.115.5.991.
Full textAbstract:
Class V myosins are one of the most ancient and widely distributed groups of the myosin superfamily and are hypothesized to function as motors for actin-dependent organelle transport. We report the discovery and initial characterization of a novel member of this family, human myosin-Vc (Myo5c). The Myo5c protein sequence shares ∼50% overall identity with the two other class V myosins in vertebrates, myosin-Va (Myo5a) and myosin-Vb (Myo5b). Systematic analysis of the mRNA and protein distribution of these myosins indicates that Myo5a is most abundant in brain, whereas Myo5b and Myo5c are expressed chiefly in non-neuronal tissues. Myo5c is particularly abundant in epithelial and glandular tissues including pancreas, prostate, mammary,stomach, colon and lung. Immunolocalization in colon and exocrine pancreas indicates that Myo5c is expressed chiefly in epithelial cells. A dominant negative approach using a GFP-Myo5c tail construct in HeLa cells reveals that the Myo5c tail selectively colocalizes with and perturbs a membrane compartment containing the transferrin receptor and rab8. Transferrin also accumulates in this compartment, suggesting that Myo5c is involved in transferrin trafficking. As a class V myosin of epithelial cells, Myo5c is likely to power actin-based membrane trafficking in many physiologically crucial tissues of the human body.
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Toepfer, Christopher N., Amanda C. Garfinkel, Gabriela Venturini, Hiroko Wakimoto, Giuliana Repetti, Lorenzo Alamo, Arun Sharma, et al. "Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy." Circulation 141, no. 10 (March 10, 2020): 828–42. http://dx.doi.org/10.1161/circulationaha.119.042339.
Full textAbstract:
Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. Methods: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Results: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.
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Ruppel, K. M., and J. A. Spudich. "Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft." Molecular Biology of the Cell 7, no. 7 (July 1996): 1123–36. http://dx.doi.org/10.1091/mbc.7.7.1123.
Full textAbstract:
We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement.
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Coluccio, L. M. "Myosin I." American Journal of Physiology-Cell Physiology 273, no. 2 (August 1, 1997): C347—C359. http://dx.doi.org/10.1152/ajpcell.1997.273.2.c347.
Full textAbstract:
The class I myosins are single-headed, actin-binding, mechanochemical “motor” proteins with heavy chains in the molecular mass range of 110-130 kDa; they do not form filaments. Each myosin I heavy chain is associated with one to six light chains that bind to specific motifs known as IQ domains. In vertebrate myosin I isoforms, the light chain is calmodulin, which is thought to regulate motor activity. Proteins similar to calmodulin are associated with myosin I isoforms from lower eukaryotes. Some myosin I isoforms from lower eukaryotes are regulated by phosphorylation; however, the phosphorylation site is not present in vertebrate myosin I isoforms. Based on sequence analyses of the amino terminal “head” domains, myosin I can be subdivided into several subclasses. Analyses of the biochemical properties of the isolated molecules and localization studies support the proposal of roles for these molecules in intracellular trafficking and changes in membrane structure. Our present understanding of the properties of these molecules and their proposed roles is reviewed here.
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Schott, Daniel H., Ruth N. Collins, and Anthony Bretscher. "Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length." Journal of Cell Biology 156, no. 1 (January 7, 2002): 35–40. http://dx.doi.org/10.1083/jcb.200110086.
Full textAbstract:
Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.
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Ma, Chenhui, Zibo Zhao, Na Wang, Muhammad Tehseen Azhar, and Xiongming Du. "Genome-Wide Identification and Comparative Analysis of Myosin Gene Family in Four Major Cotton Species." Genes 11, no. 7 (June 30, 2020): 731. http://dx.doi.org/10.3390/genes11070731.
Full textAbstract:
Myosin protein as a molecular motor, binding with Actin, plays a significant role in various physiological activities such as cell division, movement, migration, and morphology; however, there are only a few studies on plant Myosin gene family, particularly in cotton. A total of 114 Myosin genes were found in Gossypium hirsutum, Gossypium barbadense, Gossypium raimondii, and Gossypium arboreum. All Myosins could be grouped into six groups, and for each group of these genes, similar gene structures are found. Study of evolution suggested that the whole genome duplications event occurring about 13–20 MYA (millions of years ago) is the key explanation for Myosins expanse in cotton. Cis-element and qPCR analysis revealed that plant hormones such as abscisic acid, methyl jasmonate, and salicylic acid can control the expression of Myosins. This research provides useful information on the function of Myosin genes in regulating plant growth, production, and fiber elongation for further studies.
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French, Alexander R., Tobin R. Sosnick, and Ronald S. Rock. "Investigations of human myosin VI targeting using optogenetically controlled cargo loading." Proceedings of the National Academy of Sciences 114, no. 9 (February 13, 2017): E1607—E1616. http://dx.doi.org/10.1073/pnas.1614716114.
Full textAbstract:
Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.
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Sivaramakrishnan, Sivaraj, and James A. Spudich. "Coupled myosin VI motors facilitate unidirectional movement on an F-actin network." Journal of Cell Biology 187, no. 1 (September 28, 2009): 53–60. http://dx.doi.org/10.1083/jcb.200906133.
Full textAbstract:
Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.
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Wang, Kui, Zhifang Yang, Xiaohui Chen, Shunxiao Liu, Xiang Li, Liuhao Wang, Hao Yu, and Hongwei Zhang. "Characterization and analysis of myosin gene family in the whitefly (<i>Bemisia tabaci</i>)." AIMS Molecular Science 9, no. 2 (2022): 91–106. http://dx.doi.org/10.3934/molsci.2022006.
Full textAbstract:
<abstract> <p>Myosin is an actin-based motor protein that widely exists in muscle tissue and non-muscle tissue, and myosin of a diverse subfamily has obvious differences in structure and cell function. Many eukaryotes and even some unicellular organisms possess a variety of myosins. They have been well characterized in human, fungi and other organisms. However, the myosin gene family in <italic>Bemisia tabaci</italic> MEAM1 (Middle East-Asia Minor1 species) is poorly studied. In the study, we identified 15 myosin genes in <italic>B. tabaci</italic> MEAM1 based on a genome database. Myosin genes can be divided into ten classes, including subfamilies I, II, III, V, VI, VII, IX, XV, XVIII, XX in <italic>B. tabaci</italic> MEAM1. The amounts of myosin in Class I are the largest of the isoforms. Expression profiling of myosins by quantitative real-time PCR revealed that their expression differed among developmental stages and different tissues of <italic>B. tabaci</italic> MEAM1. The diversely may be related to the development characteristics of <italic>B. tabaci</italic> MEAM1. The <italic>BtaMyo-IIIb-like X1</italic> was highly expressed in nymphs 4 instar which may be related to the development process before metamorphosis. Our outcome contributes to the basis for further research on myosin gene function in <italic>B. tabaci</italic> MEAM1 and homologous myosins in other biology.</p> </abstract>
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Hoh, Joseph F. Y. "`Superfast' or masticatory myosin and the evolution of jaw-closing muscles of vertebrates." Journal of Experimental Biology 205, no. 15 (August 1, 2002): 2203–10. http://dx.doi.org/10.1242/jeb.205.15.2203.
Full textAbstract:
SUMMARY There are four fibre types in mammalian limb muscles, each expressing a different myosin isoform that finely tunes fibre mechanics and energetics for locomotion. Functional demands on jaw-closer muscles are complex and varied,and jaw muscles show considerable phylogenetic plasticity, with a repertoire for myosin expression that includes limb, developmental, α-cardiac and masticatory myosins. Masticatory myosin is a phylogenetically ancient motor with distinct light chains and heavy chains. It confers high maximal muscle force and power. It is highly jaw-specific in expression and is found in several orders of eutherian and marsupial mammals including carnivores,chiropterans, primates, dasyurids and diprotodonts. In exceptional species among these orders, masticatory myosin is replaced by some other isoform. Masticatory myosin is also found in reptiles and fish. It is postulated that masticatory myosin diverged early during gnathostome evolution and is expressed in primitive mammals. During mammalian evolution, mastication of food became important, and in some taxa jaw closers replaced masticatory myosin with α-cardiac, developmental, slow or fast limb myosins to adapt to the variety of diets and eating habits. This occurred early in some taxa(rodents, ungulates) and later in others (macropods, lesser panda, humans). The cellular basis for the uniqueness of jaw-closing muscles lies in their developmental origin.
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Rimm, D. L., J. H. Sinard, and T. D. Pollard. "Location of the head-tail junction of myosin." Journal of Cell Biology 108, no. 5 (May 1, 1989): 1783–89. http://dx.doi.org/10.1083/jcb.108.5.1783.
Full textAbstract:
The tails of double-headed myosin molecules consist of an alpha-helical/coiled-coil structure composed of two identical polypeptides with a heptad repeat of hydrophobic amino acids that starts immediately after a conserved proline near position 847. Both muscle and nonmuscle myosins have this heptad repeat and it has been assumed that proline 847 is physically located at the head-tail junction. We present two lines of evidence that this assumption is incorrect. First, we localized the binding sites of several monoclonal antibodies on Acanthamoeba myosin-II both physically, by electron microscopy, and chemically, with a series of truncated myosin-II peptides produced in bacteria. These data indicate that the head-tail junction is located near residue 900. Second, we compared the lengths of two truncated recombinant myosin-II tails with native myosin-II. The distances from the NH2 termini to the tips of these short tails confirms the rise per residue (0.148 nm/residue) and establishes that the 86-nm tail of myosin-II must start near residue 900. We propose that the first 53 residues of heptad repeat of Acanthamoeba myosin-II and other myosins are located in the heads and the proteolytic separation of S-1 from rod occurs within the heads.
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Lister, I., R. Roberts, S. Schmitz, M. Walker, J. Trinick, C. Veigel, F. Buss, and J. Kendrick-Jones. "Myosin VI: a multifunctional motor." Biochemical Society Transactions 32, no. 5 (October 26, 2004): 685–88. http://dx.doi.org/10.1042/bst0320685.
Full textAbstract:
Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed.
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Farman, Gerrie P., Priya Muthu, Katarzyna Kazmierczak, Danuta Szczesna-Cordary, and Jeffrey R. Moore. "Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on β-myosin cross-bridge mechanics." Journal of Applied Physiology 117, no. 12 (December 15, 2014): 1471–77. http://dx.doi.org/10.1152/japplphysiol.00798.2014.
Full textAbstract:
Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM.
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Müsch, Anne, David Cohen, and Enrique Rodriguez-Boulan. "Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN." Journal of Cell Biology 138, no. 2 (July 28, 1997): 291–306. http://dx.doi.org/10.1083/jcb.138.2.291.
Full textAbstract:
The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113–1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II could be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibited the release of VSVG-containing vesicles from the TGN.
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Kolega, J. "Cytoplasmic dynamics of myosin IIA and IIB: spatial ‘sorting’ of isoforms in locomoting cells." Journal of Cell Science 111, no. 15 (August 1, 1998): 2085–95. http://dx.doi.org/10.1242/jcs.111.15.2085.
Full textAbstract:
Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.
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Vivarelli, E., W. E. Brown, R. G. Whalen, and G. Cossu. "The expression of slow myosin during mammalian somitogenesis and limb bud differentiation." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2191–97. http://dx.doi.org/10.1083/jcb.107.6.2191.
Full textAbstract:
The developmental pattern of slow myosin expression has been studied in mouse embryos from the somitic stage to the period of secondary fiber formation and in myogenic cells, cultured from the same developmental stages. The results obtained, using a combination of different polyclonal and monoclonal antibodies, indicate that slow myosin is coexpressed in virtually all the cells that express embryonic (fast) myosin in somites and limb buds in vivo as well as in culture. On the contrary fetal or late myoblasts (from 15-d-old embryos) express in culture only embryonic (fast) myosin. At this stage, muscle cells in vivo, as already shown (Crow, M.T., and F.A. Stockdale. 1986. Dev. Biol. 113:238-254; Dhoot, G.K. 1986. Muscle & Nerve. 9:155-164; Draeger, A., A.G. Weeds, and R.B. Fitzsimons. 1987. J. Neurol. Sci. 81:19-43; Miller, J.B., and F.A. Stockdale. 1986. J. Cell Biol. 103:2197-2208), consist of primary myotubes, which express both myosins, and secondary myotubes, which express preferentially embryonic (fast) myosin. Under no circumstance neonatal or adult fast myosins were detected. Western blot analysis confirmed the immunocytochemical data. These results suggest that embryonic myoblasts in mammals are all committed to the mixed embryonic-(fast) slow lineage and, accordingly, all primary fibers express both myosins, whereas fetal myoblasts mostly belong to the embryonic (fast) lineage and likely generate fibers containing only embryonic (fast) myosin. The relationship with current models of avian myogenesis are discussed.
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Kulesza-Lipka, D., I. C. Baines, H. Brzeska, and E. D. Korn. "Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes." Journal of Cell Biology 115, no. 1 (October 1, 1991): 109–19. http://dx.doi.org/10.1083/jcb.115.1.109.
Full textAbstract:
The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.
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Podlubnaya, Z. A., S. L. Malyshev, K. Nieznański, and D. Stepkowski. "Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions." Acta Biochimica Polonica 47, no. 4 (December 31, 2000): 1007–17. http://dx.doi.org/10.18388/abp.2000_3954.
Full textAbstract:
In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.
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Greenberg, Michael J., Tianming Lin, Henry Shuman, and E. Michael Ostap. "Mechanochemical tuning of myosin-I by the N-terminal region." Proceedings of the National Academy of Sciences 112, no. 26 (June 8, 2015): E3337—E3344. http://dx.doi.org/10.1073/pnas.1506633112.
Full textAbstract:
Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post–power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms.
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Ueno, T., S. Yasuura, S. Watanabe, M. Hirose, T. Sekine, and T. Namihisa. "Immunocytochemical localization of myosin in rabbit liver cell." Journal of Histochemistry & Cytochemistry 36, no. 7 (July 1988): 803–6. http://dx.doi.org/10.1177/36.7.3290335.
Full textAbstract:
It has been established that the liver cell possesses its own myosin which resembles other non-muscle myosins in subunit composition and in its dependence of actin-activated Mg2+-ATPase activity on light chain phosphorylation (Ueno T, Sekine T: Biochem Int, 1987;15:1205). We have raised a specific antibody against rabbit liver cell myosin. Immunoblot analysis has shown that the purified antibody reacts only with the heavy chain of liver cell myosin. The antibody did not react with rabbit skeletal muscle myosin or with smooth muscle myosin extracted from rabbit intestinal wall. Cryostat liver sections analyzed by indirect immunofluorescence microscopy showed a characteristic polygonal staining pattern, indicating that myosin is concentrated close to the plasma membrane, particularly in the region of bile canaliculi. Myosin therefore appears to be localized in the area where actin filaments are also abundant.
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Mehta, Amit. "Myosin learns to walk." Journal of Cell Science 114, no. 11 (June 1, 2001): 1981–98. http://dx.doi.org/10.1242/jcs.114.11.1981.
Full textAbstract:
Recent experiments, drawing upon single-molecule, solution kinetic and structural techniques, have clarified our mechanistic understanding of class V myosins. The findings of the past two years can be summarized as follows: (1) Myosin V is a highly efficient processive motor, surpassing even conventional kinesin in the distance that individual molecules can traverse. (2) The kinetic scheme underlying ATP turnover resembles those of myosins I and II but with rate constants tuned to favor strong binding to actin. ADP release precedes dissociation from actin and is rate-limiting in the cycle. (3) Myosin V walks in strides averaging ∼36 nm, the long pitch pseudo-repeat of the actin helix, each step coupled to a single ATP hydrolysis. Such a unitary displacement, the largest molecular step size measured to date, is required for a processive myosin motor to follow a linear trajectory along a helical actin track.
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Yu, Hoi-Ying E., and William M. Bement. "Multiple Myosins Are Required to Coordinate Actin Assembly with Coat Compression during Compensatory Endocytosis." Molecular Biology of the Cell 18, no. 10 (October 2007): 4096–105. http://dx.doi.org/10.1091/mbc.e06-11-0993.
Full textAbstract:
Actin is involved in endocytosis in organisms ranging from yeast to mammals. In activated Xenopus eggs, exocytosing cortical granules (CGs) are surrounded by actin “coats,” which compress the exocytosing compartments, resulting in compensatory endocytosis. Here, we examined the roles of two myosins in actin coat compression. Myosin-2 is recruited to exocytosing CGs late in coat compression. Inhibition of myosin-2 slows coat compression without affecting actin assembly. This differs from phenotype induced by inhibition of actin assembly, where exocytosing CGs are trapped at the plasma membrane (PM) completely. Thus, coat compression is likely driven in part by actin assembly itself, but it requires myosin-2 for efficient completion. In contrast to myosin-2, the long-tailed myosin-1e is recruited to exocytosing CGs immediately after egg activation. Perturbation of myosin-1e results in partial actin coat assembly and induces CG collapse into the PM. Intriguingly, simultaneous inhibition of actin assembly and myosin-1e prevents CG collapse. Together, the results show that myosin-1e and myosin-2 are part of an intricate machinery that coordinates coat compression at exocytosing CGs.